Project Summary Intracellular bacterial pathogens modulate host cell functions to generate intracellular niches that promote their survival, proliferation and persistence within their host. Determining the mechanisms used to create these niches is critical to understanding their pathogenesis. Many intracellular bacteria of public health concern exploit the host cell's secretory pathway, specifically the endoplasmic reticulum (ER), to support biogenesis of their replicative niche and intracellular growth, emphasizing the importance of this organelle in pathogenesis. Bacteria of the genus Brucella, the causative agents of the worldwide zoonosis brucellosis, undergo an intracellular cycle that culminates in the biogenesis of a replication- permissive Brucella-containing vacuole (rBCV) derived from the ER, ensuring their long-term survival and proliferation. rBCV biogenesis invokes functions of the host secretory pathway at the ER interface and requires the bacterium's VirB Type IV secretion system (T4SS), via delivery of effector proteins that presumably modulate various ER functions. Despite the established role of early secretory trafficking in the Brucella infectious cycle, whether and how Brucella exploits additional ER functions remains a knowledge gap in our understanding of its pathogenesis. We have recently identified a series of T4SS effectors, among which BspA contributes to rBCV biogenesis and bacterial replication and binds the host ER-associated degradation (ERAD) E3 ubiquitin ligase MARCH6/TEB4, suggesting it impacts the ERAD pathway. Interestingly, pharmacological interference with ERAD, a central component of ER homeostasis, enhances replication of Brucella, suggesting that this pathway is deleterious to the bacterium's infectious cycle and possibly targeted by BspA. Here we will test the hypothesis that the Brucella T4SS effector BspA interacts with MARCH6 to interfere with ERAD functions and promote rBCV biogenesis and bacterial replication. First, we will use molecular and cellular approaches to elucidate the role the ERAD pathway plays in the Brucella infectious cycle and determine whether Brucella modulates it. Second, we will use various cellular and biochemical approaches to characterize the mode of action of the Brucella T4SS effector BspA on MARCH6 and elucidate the molecular basis of its contribution to rBCV biogenesis and Brucella replication. The proposed studies will likely uncover a new role of ERAD in bacterial pathogenesis, define the mode of action of a Brucella T4SS effector, and likely generate a new paradigm of pathogen interactions with the ER, a host cellular compartment essential to many intracellular microbes.